Sph2(176-191) and Sph2(446-459): Identification of B-Cell Linear Epitopes in Sphingomyelinase 2 (Sph2), Naturally Recognized by Patients Infected by Pathogenic Leptospires.

Sphingomyelin is a major constituent of eukaryotic cell membranes, and if degraded by bacteria sphingomyelinases may contribute to the pathogenesis of infection. Among Leptospira spp., there are five sphingomyelinases exclusively expressed by pathogenic leptospires, in which Sph2 is expressed during natural infections, cytotoxic, and implicated in the leptospirosis hemorrhagic complications. Considering this and the lack of information about associations between Sph2 and leptospirosis severity, we use a combination of immunoinformatics approaches to identify its B-cell epitopes, evaluate their reactivity against samples from leptospirosis patients, and investigate the role of antibodies anti-Sph2 in protection against severe leptospirosis. Two B-cell epitopes, Sph2(176-191) and Sph2(446-459), were predicted in Sph2 from L. interrogans serovar Lai, presenting different levels of identity when compared with other pathogenic leptospires. These epitopes were recognized by about 40% of studied patients with a prevalence of IgG antibodies against both Sph2(176-191) and Sph2(446-459). Remarkably, just individuals with low reactivity to Sph2(176-191) presented clinical complications, while high responders had only mild symptoms. Therefore, we identified two B-cell linear epitopes, recognized by antibodies of patients with leptospirosis, that could be further explored in the development of multi-epitope vaccines against leptospirosis.

A rare association between factor H deficiency and lupus: Case report and experimental treatment with curcumin.

Factor H (FH) is one of the most important regulatory proteins of the alternative pathway of the complement system. FH deficiency is a rare condition that causes unregulated C3 consumption, leading to an increased susceptibility to infections and glomerulopathies. Our previous studies have demonstrated a FH deficient patient carrying a c.452G > A, p.R127H FH mutation which leads to a misfolded protein and its retention in the endoplasmic reticulum. In his cultured fibroblasts, FH-delayed secretion was partially rescued when treated with curcumin, and once secreted, exhibited normal regulatory function. Here, we report a childhood-onset systemic lupus erythematosus (cSLE) in this FH deficient patient and the results of experimental treatment with curcumin aiming to rescue FH secretion and regulatory activity.

Leptolysin, a Leptospira secreted metalloprotease of the pappalysin family with broad-spectrum activity.

Extracellular proteolytic enzymes are produced by a variety of pathogenic microorganisms, and contribute to host colonization by modulating virulence. Here, we present a first characterization of leptolysin, a Leptospira metalloprotease of the pappalysin family identified in a previous exoproteomic study. Comparative molecular analysis of leptolysin with two other pappalysins from prokaryotes, ulilysin and mirolysin, reveals similarities regarding calcium, zinc, and arginine -binding sites conservation within the catalytic domain, but also discloses peculiarities. Variations observed in the primary and tertiary structures may reflect differences in primary specificities. Purified recombinant leptolysin of L. interrogans was obtained as a ~50 kDa protein. The protease exhibited maximal activity at pH 8.0 and 37°C, and hydrolytic activity was observed in the presence of different salts with maximum efficiency in NaCl. Substrate specificity was assessed using a small number of FRET peptides, and showed a marked preference for arginine residues at the P1 position. L. interrogans leptolysin proteolytic activity on proteinaceous substrates such as proteoglycans and plasma fibronectin was also evaluated. All proteins tested were efficiently degraded over time, confirming the protease´s broad-spectrum activity in vitro. In addition, leptolysin induced morphological alterations on HK-2 cells, which may be partially attributed to extracellular matrix (ECM) degradation. Hemorrhagic foci were observed in the dorsal skin of mice intradermally injected with leptolysin, as a plausible consequence of ECM disarray and vascular endothelium glycocalyx damage. Assuming that leptospiral proteases play an important role in all stages of the infectious process, characterizing their functional properties, substrates and mechanisms of action is of great importance for therapeutic purposes.

Complement System in Alcohol-Associated Liver Disease.

Innate immunity contributes effectively to the development of Alcohol-Associated liver disease (ALD). Particularly, human studies and murine models of ALD have shown that Complement activation plays an important role during the initial and later stages of ALD. The Complement System may contribute to the pathogenesis of this disease since it has been shown that ethanol-derived metabolic products activate the Complement cascade on liver membranes, leading to hepatocellular damage. However, studies evaluating the plasma levels of Complement proteins in ALD patients present contradictory results in some cases, and do not establish a well-marked role for each Complement component. The impairment of leukocyte chemoattractant activity observed in these patients may contribute to the susceptibility to bacterial infections in the latter stages of the disease. On the other hand, murine models of ALD have provided more detailed insights into the mechanisms that link the Complement System to the pathogenesis of the disease. It has been observed that Classical pathway can be activated via C1q binding to apoptotic cells in the liver and contributes to the development of hepatic inflammation. C3 contributes to the accumulation of triglycerides in the liver and in adipose tissue, while C5 seems to be involved with inflammation and liver injury after chronic ethanol consumption. In this review, we present a compendium of studies evaluating the role of Complement in human and murine models of ALD. We also discuss potential therapies to human ALD, highlighting the use of Complement inhibitors.

A double edged-sword - The Complement System during SARS-CoV-2 infection.

In the past 20 years, infections caused by coronaviruses SARS-CoV, MERS-CoV and SARS-CoV-2 have posed a threat to public health since they may cause severe acute respiratory syndrome (SARS) in humans. The Complement System is activated during viral infection, being a central protagonist of innate and acquired immunity. Here, we report some interactions between these three coronaviruses and the Complement System, highlighting the central role of C3 with the severity of these infections. Although it can be protective, its role during coronavirus infections seems to be contradictory. For example, during SARS-CoV-2 infection, Complement System can control the viral infection in asymptomatic or mild cases; however, it can also intensify local and systemic damage in some of severe COVID-19 patients, due to its potent proinflammatory effect. In this last condition, the activation of the Complement System also amplifies the cytokine storm and the pathogenicity of coronavirus infection. Experimental treatment with Complement inhibitors has been an enthusiastic field of intense investigation in search of a promising additional therapy in severe COVID-19 patients.

The Role of Properdin in Killing of Non-Pathogenic Leptospira biflexa.

Properdin (P) is a positive regulatory protein that stabilizes the C3 convertase and C5 convertase of the complement alternative pathway (AP). Several studies have suggested that properdin can bind directly to the surface of certain pathogens regardless of the presence of C3bBb. Saprophytic Leptospira are susceptible to complement-mediated killing, but the interaction of properdin with Leptospira spp. has not been evaluated so far. In this work, we demonstrate that properdin present in normal human serum, purified properdin, as well as properdin oligomers P2, P3, and P4, interact with Leptospira. Properdin can bind directly to the bacterial surface even in the absence of C3b. In line with our previous findings, AP activation was shown to be important for killing non-pathogenic L. biflexa, and properdin plays a key role in this process since this microorganism survives in P-depleted human serum and the addition of purified properdin to P-depleted human serum decreases the number of viable leptospires. A panel of pathogenic L.interrogans recombinant proteins was used to identify putative properdin targets. Lsa30, an outer membrane protein from L. interrogans, binds to unfractionated properdin and to a lesser extent to P2-P4 properdin oligomers. In conclusion, properdin plays an important role in limiting bacterial proliferation of non-pathogenic Leptospira species. Once bound to the leptospiral surface, this positive complement regulatory protein of the AP contributes to the formation of the C3 convertase on the leptospire surface even in the absence of prior addition of C3b.

Contribution of Complement System pathways to the killing of Leptospira spp.

The Complement System (CS) plays an important role in the immune response against leptospirosis and can be activated by the Alternative and Lectin Pathways (Innate Immunity) and by the Classical Pathway (Acquired Immunity). Here we analyzed a broad range of nonpathogenic and pathogenic Leptospira strains considering their interaction with each CS pathway. We determined bacterial survival rate and CS protein deposition in the presence of purified proteins, specific component depleted sera and NHS treated with the chelating agents EDTA (inhibits all three activation pathways) or EGTA (inhibits the Classical and Lectin Pathways). We suggest that the Lectin and the Alternative Pathways have an important role to eliminate saprophytic leptospires since i) approximately 50% survival of both saprophytic strains was observed in the presence of MBL-deficient serum; ii) approximately 50% survival of Leptospira biflexa Patoc I was observed in the presence of NHS - EGTA and iii) C1q-depleted serum caused significant bacterial lysis. In all serovars investigated the deposition of C5-C9 proteins on saprophytic Leptospira strains was more pronounced when compared to pathogenic species confirming previous studies in the literature. No difference on C3 deposition was observed between nonpathogenic and pathogenic strains. In conclusion, Leptospira strains interact to different degrees with CS proteins, especially those necessary to form MAC, indicating that some strains and specific ligands could favor the binding of certain CS proteins.

Complement Resistance Assays.

Like many other pathogens of medical importance, pathogenic Leptospira employ diverse strategies to circumvent Complement System activation. Under physiological conditions, this central humoral arm of innate immunity is tightly controlled by negative Complement regulatory proteins. However, upon infection, pathogenic microorganisms interfere with normal Complement host defense mechanisms by recruiting or mimicking Complement regulators and by secreting endogenous proteases or acquiring host's proteases that inactivate key Complement components. In this chapter, we describe in detail some of the most frequently used assays to evaluate Leptospira Complement resistance.

Strategies used by Leptospira spirochetes to evade the host complement system.

Leptospires are highly invasive spirochetes equipped with efficient strategies for dissemination in the host. The Leptospira genus currently comprises 64 species divided into two major clades: the saprophytes composed of nonpathogenic, free-living organisms, and the pathogens encompassing all the species that cause mild or severe infections in humans and animals. While saprophytes are highly susceptible to the lytic action of the complement system, pathogenic (virulent) strains have evolved virulence strategies that allow efficient colonization of a variety of hosts and target organs, including mechanisms to circumvent hosts' innate and acquired immune responses. Pathogenic Leptospira avoid complement-mediated killing by recruiting host complement regulatory proteins and by targeting complement proteins using own and host-expressed proteases. This review outlines the role of complement in eradicating saprophytic Leptospira and the stratagems adopted by pathogenic Leptospira to maneuver the host complement system for their benefit.

Cytokine Profile in Early Infection by Leptospira interrogans in A/J Mice.

Leptospirosis is considered a neglected disease with an estimated more than one million cases every year. Since rodents are at the same time the main reservoir and generally asymptomatic to Leptospira infection, understanding why some animal species are resistant and others are susceptible to this infection would shed some light in how to control this important zoonosis. The innate immune response against Leptospira is mainly dependent on phagocytosis and activation of the Complement System. In this context, cytokines may drive the early control of infection and the adaptive response. Since the Complement System is important to eliminate leptospires in vivo, we investigated if Complement C5 in A/J mice would modulate the cytokine production during infection by Leptospira interrogans serovar Kennewicki type Pomona Fromm (LPF). Thus, our aim was to investigate the systemic levels of pro- and anti-inflammatory cytokines during Leptospira infection in the blood, liver, lung, and kidney on the third and sixth days of infection in A/J C5+/+ and A/J C5-/- mice. Blood levels of TNF-α, IL-6, IFN-γ, and MCP-1 reached a peak on the third day. Although both mouse strains developed splenomegaly, similar histopathological alterations in the liver and the lung, levels of pro- and anti-inflammatory cytokines were different. A/J C5+/+ mice had higher levels of liver IL-10, IL-1ß, IL-12p40, and IL-12p70 and kidney IL-1ß, IL-12p40, and IL-12p70 on the sixth day of infection when compared to A/J C5-/- mice. Our results showed that in A/J genetic background, the Complement component C5 modulates a cytokine profile in the liver and kidney of infected mice, which may play a role in the control of disease progression.

Culture-attenuated pathogenic Leptospira lose the ability to survive to complement-mediated-killing due to lower expression of factor H binding proteins.

Several pathogens including Gram-negative bacteria hijack complement regulators to escape host's innate response. Pathogenic Leptospira species bind Factor H, C4b binding protein and vitronectin from the complement system. We evaluated the ability of low passage (LP) and culture-attenuated (CA) pathogenic strains of Leptospira, to bind Factor H. We used LOCaS46 (Leptospira interrogans sv Canicola), LOVe30 (L. interrogans sv Icterohaemorrhagiae) and MOCA45 (L. santarosai sv Tarassovi), and ten high passage strains of Leptospira [used in the microscopic agglutination test (MAT)]. Afterwards, we assessed their survival in normal human serum (NHS). Interestingly, the ability in binding Factor H was higher for LOCaS46 and LOVe30 LP strains, than for the respective CA strains suggesting that the ability of evading the alternative complement pathway is lost after culture attenuation. Accordingly, the level of mRNA expression of the Factor H binding proteins, LigA, LigB and Lsa23 was higher in these LP strains than in the corresponding CA strains. Unexpectedly, no difference in Factor H binding and surviving was observed between LP and CA MOCA45 strains. The high passage MAT-reference strains showed variation in Factor H binding ability, but, in most cases, the ability for capturing Factor H by Leptospira strains correlated with their survival in NHS.

Leptospira interrogans thermolysin refolded at high pressure and alkaline pH displays proteolytic activity against complement C3.

Enzymes from the thermolysin family are crucial factors in the pathogenesis of several diseases caused by bacteria and are potential targets for therapeutic interventions. Thermolysin encoded by the gene LIC13322 of the causative agent of leptospirosis, Leptospira interrogans, was shown to cleave proteins from the Complement System. However, the production of this recombinant protein using traditional refolding processes with high levels of denaturing reagents for thermolysin inclusion bodies (TL-IBs) solubilization results in poor recovery and low proteolytic activity probably due to improper refolding of the protein. Based on the assumption that leptospiral proteases play a crucial role during infection, the aim of this work was to obtain a functional recombinant thermolysin for future studies on the role of these metalloproteases on leptospiral infection. The association of high hydrostatic pressure (HHP) and alkaline pH was utilized for thermolysin refolding. Incubation of a suspension of TL-IBs at HHP and a pH of 11.0 is non-denaturing but effective for thermolysin solubilization. Soluble protein does not reaggregate by dialysis to pH 8.0. A volumetric yield of 46 mg thermolysin/L of bacterial culture and a yield of near 100% in relation to the total thermolysin present in TL-IBs were obtained. SEC-purified thermolysin suffers fragmentation, likely due to autoproteolysis and presents proteolytic activity against complement C3 α-chain, possibly by a generation of a C3b-like molecule. The proteolytic activity of thermolysin against C3 was time and dose-dependent. The experience gained in this study shall help to establish efficient HHP-based processes for refolding of bioactive proteins from IBs.

Leptospira interrogans Secreted Proteases Degrade Extracellular Matrix and Plasma Proteins From the Host.

Leptospires are highly motile spirochetes equipped with strategies for efficient invasion and dissemination within the host. Our group previously demonstrated that pathogenic leptospires secrete proteases capable of cleaving and inactivating key molecules of the complement system, allowing these bacteria to circumvent host's innate immune defense mechanisms. Given the successful dissemination of leptospires during infection, we wondered if such proteases would target a broader range of host molecules. In the present study, the proteolytic activity of secreted leptospiral proteases against a panel of extracellular matrix (ECM) and plasma proteins was assessed. The culture supernatant of the virulent L. interrogans serovar Kennewicki strain Fromm (LPF) degraded human fibrinogen, plasma fibronectin, gelatin, and the proteoglycans decorin, biglycan, and lumican. Interestingly, human plasminogen was not cleaved by proteases present in the supernatants. Proteolytic activity was inhibited by 1,10-phenanthroline, suggesting the participation of metalloproteases. Moreover, production of proteases might be an important virulence determinant since culture-attenuated or saprophytic Leptospira did not display proteolytic activity against ECM or plasma components. Exoproteomic analysis allowed the identification of three metalloproteases that could be involved in the degradation of host components. The ability to cleave conjunctive tissue molecules and coagulation cascade proteins may certainly contribute to invasion and tissue destruction observed upon infection with Leptospira.

Role of Murine Complement Component C5 in Acute in Vivo Infection by Pathogenic Leptospira interrogans.

Leptospirosis is considered one of the most important zoonosis worldwide. The activation of the Complement System is important to control dissemination of several pathogens in the host. Only a few studies have employed murine models to investigate leptospiral infection and our aim in this work was to investigate the role of murine C5 during in vivo infection, comparing wild type C57BL/6 (B6 C5+/+) and congenic C57BL/6 (B6 C5-/-, C5 deficient) mice during the first days of infection. All animals from both groups survived for at least 8 days post-infection with pathogenic Leptospira interrogans serovar Kennewicki strain Fromm (LPF). At the third day of infection, we observed greater numbers of LPF in the liver of B6 C5-/- mice when compared to B6 C5+/+ mice. Later, on the sixth day of infection, the LPF population fell to undetectable levels in the livers of both groups of mice. On the third day, the inflammatory score was higher in the liver of B6 C5+/+ mice than in B6 C5-/- mice, and returned to normal on the sixth day of infection in both groups. No significant histopathological differences were observed in the lung, kidney and spleen from both infected B6 C5+/+ than B6 C5-/- mice. Likewise, the total number of circulating leukocytes was not affected by the absence of C5. The liver levels of IL-10 on the sixth day of infection was lower in the absence of C5 when compared to wild type mice. No significant differences were observed in the levels of several inflammatory cytokines when B6 C5+/+ and B6 C5-/- were compared. In conclusion, C5 may contribute to the direct killing of LPF in the first days of infection in C57BL/6 mice. On the other hand, other effector immune mechanisms probably compensate Complement impairment since the mice survival was not affected by the absence of C5 and its activated fragments, at least in the early stage of this infection.

Complement Immune Evasion by Spirochetes.

The complement system plays an important role in the innate and acquired immune response against pathogens. A sophisticated network of activating and regulating proteins allows the distinction between intact and damaged host and non-host surfaces such as bacteria and other parasites. Non-host structures trigger the alternative pathway which may lead to their elimination by phagocytosis or cell lysis. In addition, complement proteins such as C1q, mannose binding lectin (MBL), and ficolins act as pathogen pattern-recognition molecules. Biological functions such as opsonization, activation of B lymphocytes and production of antibodies, degranulation of mast cells and basophils, and cell lysis that are important for elimination of microorganisms are dependent on complement activation. However, several pathogens including spirochetes have developed several specialized mechanisms to evade the complement system, thereby contributing to survival in the host. In this review, we give a brief overview of complement activation and regulation, and discuss in detail the strategies used by spirochetes from the genera Borrelia, Leptospira, and Treponema to overcome complement activation.

Binding of human complement C1 sterase inhibitor to Leptospira spp.

Leptospirosis is an important zoonosis of global importance caused by bacteria Leptospira spp. Pathogenic Leptospira is resistant to Complement System killing while non-pathogenic Leptospira is rapidly killed by exposure to normal human serum (NHS). Pathogenic Leptospira interact with Complement Regulators such as Factor H, C4b binding protein and Vitronectin avoiding Complement activation and killing by Alternative and Classical Pathways. One important regulator is C1-inhibitor (C1INH) that interacts with C1s or MASPs controlling the cleavage of C4 and C2 molecules, thereby inhibiting the activation of the Classical and Lectin Pathways. In this study, we demonstrate that attenuated, saprophytic and pathogenic Leptospira interact with C1INH that maintain its regulatory capacity of interaction with C1s preventing the activation of Complement system. Although the interaction with C1INH is not crucial for pathogenic Leptospira survival, it seems to be important for the survival of attenuated and saprophytic Leptospira in normal human serum.

Pathogenic Leptospira Secreted Proteases Target the Membrane Attack Complex: A Potential Role for Thermolysin in Complement Inhibition.

Leptospirosis is a zoonosis caused by spirochetes from the genus Leptospira. This disease is common in tropical and subtropical areas, constituting a serious public health problem. Pathogenic Leptospira have the ability to escape the human Complement System, being able to survive when in contact with normal human serum. In a previous study, our group demonstrated that supernatants of pathogenic Leptospira (SPL) inhibit the three activation pathways of the Complement System. This inhibition can be directly correlated with the activity of secreted proteases, which cleave the Complement molecules C3, Factor B (Alternative Pathway), C4 and C2 (Classical and Lectin Pathways). In this work, we analyze the activity of the leptospiral proteases on the components of Terminal Pathway of Complement, called the membrane attack complex (MAC). We observed that proteases present in SPL from different Leptospira strains were able to cleave the purified proteins C5, C6, C7, C8, and C9, while culture supernatant from non-pathogenic Leptospira strains (SNPL) had no significant proteolytic activity on these substrates. The cleavages occurred in a time-dependent and specificity manner. No cleavage was observed when we used whole serum as a source of C5-C9 proteins, probably because of the abundant presence of plasma protease inhibitors such as α2-macroglobulin. Complement protein cleavage by SPL was inhibited by 1,10-phenanthroline, indicating the involvement of metalloproteases. Furthermore, 1,10-phenanthroline- treated normal human serum diminished pathogenic leptospira survival. We also analyzed the proteolytic activity of thermolysin (LIC13322) a metalloprotease expressed exclusively by pathogenic Leptospira strains. Recombinant thermolysin was capable of cleaving the component C6, either purified or as part of the SC5b-9 complex. Furthermore, we found that the MAC proteins C6-C9 interact with thermolysin, indicating that this metalloprotease may have an additional inhibitory effect on these molecules by direct interactions. Finally, a functional assay demonstrated that thermolysin was able to inhibit MAC-dependent erythrocytes lysis. We conclude that proteases secreted exclusively by pathogenic Leptospira strains are capable of degrading several Complement effector molecules, representing potential targets for the development of new therapies and prophylactic approaches in leptospirosis.

The complement component C5 promotes liver steatosis and inflammation in murine non-alcoholic liver disease model.

Non-Alcoholic Fatty Liver Disease (NALD) is considering a hepatic manifestation of metabolic syndrome. Although the pathogenesis of NALD is not completely understood, insulin resistance and inflammatory cytokines are implicated. Considering that component C5 is a central mediator of inflammation, we investigated the role of C5 in the establishment of NALD. Eight to ten-week old B6 C5(+) and A/J C5(-) male mice were fed a high fat diet containing glucose (HFDG) for 6 and 10 weeks. We observed that B6 C5(+) mice HFDG-fed for 10 weeks developed hepatomegaly, triglycerides (TG) accumulation, steatosis and enhanced liver TNF-α, IL-6, IL-12p70 and IL-17 levels when compared to A/J C5(-) mice. Next, B6 C5(+) mice were compared with congenic B6 C5(-) mice. Again, B6 C5(+) HFDG-fed mice developed more steatosis, liver centro-lobular inflammation and presented higher levels of liver IL-1ß, IL-12p70, IL-17 and TFG-ß than B6 C5(-) mice under the same conditions. B6 C5(+) mice HFDG-fed also presented lower concentrations of serum albumin, serum cholesterol, blood leukocytes and liver NO production when compared with B6 C5(-) mice. We concluded that murine C5 contributes effectively to liver steatosis and inflammation in NALD pathogenesis. In addition, C5 is also important to control serum cholesterol and albumin levels in the C57BL/6 genetic background.

Acquisition of negative complement regulators by the saprophyte Leptospira biflexa expressing LigA or LigB confers enhanced survival in human serum.

Leptospiral immunoglobulin-like (Lig) proteins are surface exposed molecules present in pathogenic but not in saprophytic Leptospira species. We have previously shown that Lig proteins interact with the soluble complement regulators Factor H (FH), FH like-1 (FHL-1), FH related-1 (FHR-1) and C4b Binding Protein (C4BP). In this study, we used the saprophyte L. biflexa serovar Patoc as a surrogate host to address the specific role of LigA and LigB proteins in leptospiral complement evasion. L. biflexa expressing LigA or LigB was able to acquire FH and C4BP. Bound complement regulators retained their cofactor activities of FI in the proteolytic cleavage of C3b and C4b. Moreover, heterologous expression of ligA and ligB genes in the saprophyte L. biflexa enhanced bacterial survival in human serum. Complement deposition on lig-transformed L. biflexa was assessed by flow cytometry analysis. With regard to MAC deposition, L. biflexa expressing LigA or LigB presented an intermediate profile: MAC deposition levels were greater than those found in the pathogenic L. interrogans, but lower than those observed for L. biflexa wildtype. In conclusion, Lig proteins contribute to in vitro control of complement activation on the leptospiral surface, promoting an increased bacterial survival in human serum.